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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
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  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
  • C12N9/2411—Amylases
  • C12N9/2414—Alpha-amylase (3.2.1.1.)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
  • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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  • C12N2330/00—Production
  • C12N2330/50—Biochemical production, i.e. in a transformed host cell
  • C12N2330/51—Specially adapted vectors
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  • C12N2510/00—Genetically modified cells
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  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus

Definitions

• the invention is in the field of biotechnology, in particular microbial protein synthesis. More particularly, the invention relates to a method of producing proteins by genetically modified microorganisms and further proposing microorganisms used in such methods. The invention further relates to uses of such microorganisms for protein production.
• Recyclable materials are, for example, low molecular weight compounds, such as food supplements or pharmaceutically active compounds, or proteins, for which, due to their diversity, in turn, there is a large technical field of application.
• the metabolic properties of the microorganisms in question are utilized and / or modified for the production of valuable substances; in the second case, preferably microorganisms are used which express the genes of the proteins of interest.
• biotechnological production of the relevant microorganisms are cultured in fermenters, which are designed according to the metabolic properties of the microorganisms.
• the microorganisms metabolize the substrate offered and form the desired product which, after completion of the fermentation, is usually separated from the production organisms and purified and / or concentrated from the fermenter slurry and / or the fermentation medium.
• complex protein rich raw materials are typically used as a substrate besides a carbon source (typically glucose).
• the protein production thus corresponds to a biotransformation of substrate protein to the target protein. This requires complete hydrolysis of the substrate protein into the individual amino acids, which are then available for biosynthesis of the target protein.
• bacteria in microbial fermentations is basically desirable. Bacteria are characterized by short generation times and low demands on cultivation conditions. As a result, inexpensive cultivation methods or production methods can be established. In addition, the expert has bacteria in the Fermentation technology over a wealth of experience. Grampositive bacteria are preferably used because they secrete the protein to be produced (target protein) into the surrounding medium.
• the international patent application discloses WO 91/02792 the improved fermentative production of an alkaline protease from Bacillus lentus in an optimized Bacillus licheniformis strain under the control of gene regulatory sequences from Bacillus licheniformis, in particular the Bacillus licheniformis promoter.
• Feng Y. et al. discloses a genetically modified B. pumilus strain expressing amylase.
• the object of the present invention is to achieve a high product yield, in particular of a protein, in a microbial fermentation.
• a process according to the invention is consequently a fermentation process.
• Bacillus pumilus As a production organism , an advantageous, in particular increased, product yield can be achieved.
• Bacillus licheniformis a production organism established in the prior art which is used industrially in a large number of microbial fermentations.
• the method according to the invention is consequently a method for increasing the expression of a protein in a microorganism.
• An increased expression of the protein is present when a larger amount of protein is obtained by a method according to the invention in comparison with a similar method, which differs from a method according to the invention only in that bacteria of the species Bacillus licheniformis, preferably of the wild type, are used , Both methods to be compared are carried out in this respect under the same conditions as optimal as possible for the microorganisms and for the same duration.
• An expression construct is a nucleic acid sequence that causes the protein to be expressed in the microorganism. It comprises the genetic information, ie the nucleic acid sequence (gene) which codes for the protein.
• the expression of a nucleic acid sequence is its translation into the gene product (s) encoded by this sequence, ie into a polypeptide (protein) or into a plurality of polypeptides (proteins).
• polypeptide and protein are used synonymously in the present application. For the purposes of the present invention, expression thus refers to the biosynthesis of ribonucleic acid (RNA) and proteins from the genetic information.
• the expression comprises the transcription, ie the synthesis of messenger ribonucleic acid (mRNA) on the basis of the DNA (deoxyribonucleic acid) sequence of the gene and its translation into the corresponding polypeptide chain, which may be post-translationally modified can.
• mRNA messenger ribonucleic acid
• the expression of a protein thus describes its biosynthesis from the genetic information present in the microorganism according to the invention.
• An expression construct further comprises at least one nucleic acid sequence, preferably DNA, with a control function for the expression of the nucleic acid sequence coding for the protein or the auxiliary protease (so-called gene regulatory sequence).
• a gene regulatory sequence here is any nucleic acid sequence whose presence in the microorganism influences, preferably increases, the transcription frequency of the nucleic acid sequence which codes for the protein. It is preferably a promoter sequence, since such a sequence is essential for the expression of a nucleic acid sequence.
• an expression construct according to the invention may also comprise further gene regulatory sequences, for example, one or more enhancer sequences.
• An expression construct in the context of the invention thus comprises at least one functional unit of gene and promoter. It can, but does not necessarily have to, exist as a physical entity.
• a promoter is understood as meaning a DNA sequence which enables the regulated expression of a gene.
• a promoter sequence is part of a gene and is often at its 5 'end, and thus before the RNA coding region.
• the promoter sequence in an expression construct according to the invention is 5'-downstream of the nucleic acid sequence coding for the protein.
• the most important property of a promoter is the specific interaction with at least one DNA-binding protein or polypeptide, which mediates the start of transcription of the gene by an RNA polymerase and is referred to as a transcription factor.
• a promoter is therefore preferably a DNA sequence with promoter activity, i. a DNA sequence to which at least one transcription factor binds, at least transiently, to initiate transcription of a gene.
• the strength of a promoter is measurable via the transcription frequency of the expressed gene, ie via the number of RNA molecules generated per unit time, in particular mRNA molecules.
• a promoter of an expression construct according to the invention may be a promoter of the microorganism. Such a promoter sequence is thus naturally present in the microorganism.
• a promoter of an expression construct according to the invention may also have been recombinantly introduced into the microorganism. The same applies to all other gene regulatory sequences which an expression construct according to the invention may have.
• the promoter in an expression construct as used in a method according to the invention effects the expression of the nucleic acid sequence coding for the protein (target protein) in the expression construct.
• the promoter has a nucleic acid sequence as described above.
• the promoter is characterized in that it contains at least 80%, and more preferably at least 81.0%, 82.0%, 83.0%, 84.0 of the nucleic acid sequence corresponding to the nucleic acid sequence given in SEQ ID NO: 1 %, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0 %, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.2%, 99.3%, 99.4%, 99.5%, and most preferably is 100% identical, and the promoter causes a transcription frequency of the gene expressed by it, which corresponds at least to that of a promoter according to SEQ ID NO: 1.
• the promoter is characterized by having a nucleic acid sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 2 of at least 80%, and more preferably at least 81.0%, 82.0%, 83.0%, 84.0 %, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0 %, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.2%, 99.3%, 99.4%, 99.5%, and most preferably is 100% identical, and the promoter causes a transcription frequency of the gene expressed by it, which corresponds at least to that of a promoter according to SEQ ID NO: 2.
• the promoter is characterized in that it has a nucleic acid sequence which is at least 80% and more preferably at least 81.0%, 82.0%, 83.0%, of the nucleic acid sequence given in SEQ ID NO: 3.
• the promoter causes a transcription frequency of the gene expressed by it, which corresponds at least to that of a promoter according to SEQ ID NO: 3.
• the promoter is characterized by having a nucleic acid sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 4 at least 80%, and more preferably at least 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89 , 0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, 99 , 0%, 99.2%, 99.3%, 99.4%, 99.5%, and most preferably 100% identical, and the promoter causes a transcription frequency of the gene expressed by it which is at least that of a promoter according to SEQ ID NO: 4 corresponds.
• the promoter has a nucleic acid sequence as described above.
• nucleic acid or amino acid sequences is determined by a sequence comparison. Such a comparison is made by assigning similar sequences in the nucleotide sequences or amino acid sequences to each other. This sequence comparison is preferably carried out based on the BLAST algorithm established and commonly used in the prior art (cf., for example, US Pat Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool.” J. Mol. Biol. 215: 403-410 , and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J.
• T-Coffee cf., for example Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217 ) or programs based on these programs or algorithms.
• sequence comparisons and alignments are preferably created using the Vector NTI® Suite 10.3 computer program (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the default default parameters specified.
• sequence matches can be useful to relate sequence matches only to individual, possibly small areas. Unless stated otherwise, identity or homology information in the present application, however, refers to the total length of the respectively indicated nucleic acid or amino acid sequence. In the case of proteins, in particular enzymes and, in particular, proteases, the data also refer to the mature protein, unless stated otherwise. Unless otherwise stated, a sequence analysis of a protein is always directed to the mature, ready-processed protein, even if the immature form encoded by the associated gene is still processed to the mature form after translation.
• the expression construct to be introduced into the microorganism in a method according to the invention further codes for a protein. It thus comprises a nucleic acid sequence encoding this protein.
• a nucleic acid sequence encoding this protein.
• any nucleic acid sequence which can be translated into a protein can be used for this purpose.
• This is the protein which is to be produced by means of a method according to the invention (target protein).
• target protein Preferably, it is an enzyme, more preferably an enzyme as described below.
• Nucleic acids and expression constructs according to the invention can be produced by methods known per se for the modification of nucleic acids. Such are for example in relevant manuals like that of Fritsch, Sambrook and Maniatis, "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, New York, 1989 , illustrated and familiar to those skilled in the field of biotechnology. Examples of such methods are the chemical synthesis or the polymerase chain reaction (PCR), optionally in conjunction with other molecular biological and / or chemical or biochemical standard methods.
• PCR polymerase chain reaction
• the present invention is particularly suitable for the recombinant production of proteins, in particular enzymes.
• the expression construct is introduced into the microorganism, preferably by transformation.
• the introduction of the respective expression construct or parts thereof preferably takes place via vectors, in particular expression vectors.
• only parts of the expression construct, preferably at least the nucleic acid which codes for the protein are introduced into the microorganism in such a way that the finished expression construct first arises in the microorganism.
• the concept of introduction thus includes the possibility that an expression construct is completely introduced into the microorganism, preferably transformed, but also the possibility that only a part of the expression construct, particularly preferably the nucleic acid coding for the protein, is introduced into the microorganism , preferably transformed, and the complete expression construct is formed only in the microorganism. However, at least part of the expression construct is always introduced into the microorganism within the scope of the invention.
• Vectors are known to those skilled in the biotechnology field. Especially when used in bacteria, they are special plasmids, ie circular genetic elements.
• the expression construct is preferably cloned into a vector.
• the vectors may include, for example, those derived from bacterial plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements of various origins.
• vectors are able to establish themselves as stable units in the microorganisms over several generations. For the purposes of the invention, it is irrelevant whether they establish themselves as extrachomosomal units or integrate into the chromosomal DNA. Which of the numerous systems is chosen depends on the individual case. Decisive factors can be, for example, the achievable copy number, the available selection systems, including, in particular, antibiotic resistances, or the cultivability of the microorganisms capable of accepting the vectors.
• Expression vectors may also be regulatable by changes in culture conditions, such as cell density or the addition of certain compounds.
• An example of such a compound is the galactose derivative isopropyl- ⁇ -D-thiogalactopyranoside (IPTG), which is used as the activator of the bacterial lactose operon (lac operon).
• a method according to the invention is characterized in that the protein is not naturally present in the microorganism.
• the protein is not a separate protein or enzyme of the microorganism.
• the protein can therefore be found in the Microorganism are not expressed by a nucleic acid sequence which is part of the chromosomal DNA of the microorganism in its wild-type form.
• the protein and / or the nucleic acid sequence coding therefor is therefore not present in the wild-type form of the microorganism and / or can not be isolated from the wild-type form of the microorganism.
• a protein not naturally present in the microorganism or the nucleic acid sequence coding therefor has been deliberately introduced into the microorganism with the aid of genetic engineering, so that the microorganism has been enriched by the protein or the nucleic acid sequence coding therefor.
• one protein may quite naturally be present in another microorganism - relevant for the consideration is exclusively the microorganism used in the process.
• the method is characterized in that the protein is an enzyme, in particular an acid cellulase, alpha-amylase, alpha-acetodecarboxylase, aminopetidase, amylase, arabanase, beta-glucanase, beta-glucosidase, beta-mannosidase, Carageenase, carbohydrate, catalase, cellobiose oxidase, cellulase, chymosin, endo-1,3-beta-glucanase, endo-1,3 (4) -beta-glucanase, endo-1,4-beta-xylanase, endopeptidase, esterase , Exopeptidase, G4-amylase, glucoamylase, glucose oxidase, glucosidase, glycolipase, hemicellulase, laccase, lipase, lysophosphose, glucoamy
• the protein is a protease.
• the enzymes mentioned below can be advantageously prepared.
• subtilisins are preferred. Examples thereof are the subtilisins BPN 'and Carlsberg, the protease PB92, the subtilisins 147 and 309, the alkaline protease from Bacillus lentus, subtilisin DY and the enzymes thermitase, proteinase K and the subtilases, but not the subtilisins in the narrower sense Proteases TW3 and TW7.
• Subtilisin Carlsberg is available in a further developed form under the trade name Alcalase® from Novozymes A / S, Bagsvaerd, Denmark.
• subtilisins 147 and 309 are among the Trade name Esperase®, or Savinase® sold by the company Novozymes. From the protease from Bacillus lentus DSM 5483 derived under the name BLAP® protease variants derived. Further preferred proteases are, for example, the enzymes known as PUR.
• proteases include those under the trade names Durazym®, Relase®, Everlase®, Nafizym®, Natalase®, Kannase® and Ovozyme® from Novozymes under the trade names, Purafect®, Purafect® OxP, Purafect® Prime, Excellase ® and Properase® from Genencor / Danisco, sold under the trade name Protosol® by Advanced Biochemicals Ltd., Thane, India, under the trade name Wuxi® by Wuxi Snyder Bioproducts Ltd., China, under the trade names Proleather® and Protease P® from Amano Pharmaceuticals Ltd., Nagoya, Japan, and the enzyme available under the name Proteinase K-16 from Kao Corp., Tokyo, Japan. Also preferred are the proteases from Bacillus gibsonii and Bacillus pumilus, which are disclosed in the international patent applications WO2008 / 086916 and WO2007 / 131656 ,
• amylases are the ⁇ -amylases from Bacillus licheniformis, from Bacillus amyloliquefaciens or from Bacillus stearothermophilus and, in particular, also their improved developments for use in detergents or cleaners.
• the enzyme from Bacillus licheniformis is available from the company Novozymes under the name Termamyl® and from the company Danisco / Genencor under the name Purastar®ST.
• this ⁇ -amylase is available from the company Novozymes under the trade name Duramyl® and Termamyt®ultra, from the company Danisco / Genencor under the name Purastar®OxAm and from the company Daiwa Seiko Inc., Tokyo, Japan, as Keistase®.
• the Bacillus amyloliquefaciens ⁇ -amylase is sold by the company Novozymes under the name BAN®, and variants derived from the Bacillus stearothermophilus ⁇ -amylase under the names BSG® and Novamyl®, also from the company Novozymes.
• the ⁇ -amylase from Bacillus sp.
• a 7-7 (DSM 12368) and cyclodextrin glucanotransferase (CGTase) from Bacillus agaradherens (DSM 9948).
• CCTase cyclodextrin glucanotransferase
• fusion products of all the molecules mentioned can be used.
• the further developments of the ⁇ -amylase from Aspergillus niger and A. oryzae available under the trade name Fungamyl® from the company Novozymes are suitable.
• Further advantageous commercial products are, for example, the Amylase Powerase® from the company Danisco / Genencor and the amylases Amylase-LT®, Stainzyme® and Stainzyme Plus®, the latter from the company Novozymes.
• Amylases to be prepared according to the invention are also preferably ⁇ -amylases.
• lipases or cutinases are the lipases which were originally obtainable from Humicola lanuginosa (Thermomyces lanuginosus) or further developed, in particular those with the amino acid exchange D96L. They are sold for example by the company Novozymes under the trade names Lipolase®, Lipolase®Ultra, LipoPrime®, Lipozyme® and Lipex®. Furthermore, for example, the cutinases can be produced, which were originally isolated from Fusarium solani pisi and Humicola insolens .
• the lipases or cutinases can be produced, the initial enzymes were originally isolated from Pseudomonas mendocina and Fusarium solanii .
• Further important commercial products are the preparations M1 Lipase® and Lipomax® which were originally sold by Gist-Brocades (now Danisco / Genencor) and those from Meito Sangyo KK, Japan, under the name Lipase MY-30®, Lipase OF® and Lipase PL® distributed enzymes, also the product Lumafast® from Danisco / Genencor.
• cellulases examples include sequences of the fungal, endoglucanase (EG) -rich cellulase preparation or their further developments, which is offered by the company Novozymes under the trade name Celluzyme®. Endolase® and Carezyme®, also available from Novozymes, are based on the 50 kD EG or 43 kD EG from Humicola insolens DSM 1800. Further manufacturable commercial products of this company are Cellusoft®, Renozyme® and Celluclean®.
• Also manufacturable are, for example, cellulases available from AB Enzymes, Finland, under the tradenames Ecostone® and Biotouch®, which are based, at least in part, on the 20 kD EG of melanocarpus.
• Other cellulases from AB Enzymes are Econase® and Ecopulp®.
• Other suitable cellulases are from Bacillus sp. CBS 670.93 and CBS 669.93, those derived from Bacillus sp. CBS 670.93 is available from the company Danisco / Genencor under the trade name Puradax®.
• Other manufacturable commercial products of Danisco / Genencor are "Genencor detergent cellulase L" and lndiAge®Neutra.
• variants of these enzymes obtainable by point mutations can be prepared according to the invention.
• Particularly preferred cellulases are Thielavia terrestris cellulase variants described in International Publication WO 98/12307 Cellulases from Melanocarpus, in particular Melanocarpus albomyces, disclosed in the international publication WO 97/14804
• Cellulases of the EGIII type from Trichoderma reesei disclosed in the European patent application EP 1305432 are disclosed or available variants thereof, in particular those disclosed in the European patent applications EP 1240525 and EP 1305432 , as well as cellulases, which are disclosed in international publications WO 1992006165 .
• WO 96/29397 and WO 02/099091 Their respective disclosure is therefore expressly referred to.
• Suitable enzymes for this purpose are, for example, under the names Gamanase®, Pektinex AR® and Pectaway® from Novozymes, under the name Rohapec® B1L from AB Enzymes and under the name Pyrolase® from Diversa Corp., San Diego, CA. , USA available.
• the ⁇ -glucanase obtained from Bacillus subtilis is available under the name Cereflo® from Novozymes.
• Hemicellulases which are particularly preferred according to the invention are mannanases which are sold, for example, under the trade names Mannaway® by the company Novozymes or Purabrite® by the company Genencor.
• oxidoreductases for example oxidases, oxygenases, catalases, peroxidases, such as halo-, chloro-, bromo-, lignin, glucose or manganese peroxidases, dioxygenases or laccases (phenol oxidases, polyphenol oxidases) can also be prepared.
• Suitable commercial products are Denilite® 1 and 2 from Novozymes.
• Other enzymes are in international patent applications WO 98/45398 .
• WO 2005/056782 WO 2004/058961 such as WO 2005/124012 disclosed.
• the method is characterized in that the microorganism is Bacillus pumilus DSM 14395.
• This strain was deposited with the DSMZ (DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffen No 7 B, 38124 Braunschweig, Germany) on 01.03.2001 and is identified by the DSMZ as a Bacillus pumilus strain (DSM ID 01-197). As the examples in the present application show, this strain achieves very good product yields in microbial fermentations.
• the strain to be used in a method according to the invention is genetically modified.
• the genetic modification advantageously further increases the product yield or advantageously alters a property of the product.
• the product here is the expressed protein which is present in the fermentation medium. For example, its odor is reduced, its color is attenuated and / or clarify the product (ie less cloudy) or reduce its density.
• the genetic modification in this regard does not mean the introduction of the expression construct according to method step (a). Rather, the microorganism of the species Bacillus pumilus to be used in the method according to the invention is already genetically modified, specifically before the expression construct according to method step (a) is introduced into the microorganism.
• the presence of the genetic modification is determined in comparison with the Bacillus pumilus wild-type, in particular Bacillus pumilus DSM 14395.
• the genetic modification causes the functional change, for example the functional inactivation, of a gene in the microorganism.
• the functional change, for example the functional inactivation, of the gene then in turn results in an increased and / or improved production of the protein and thus an improved product yield and / or the obtaining of a product with one or more improved properties.
• functional inactivation is meant that the gene product (s) encoded by this gene are no longer formed or formed in a biologically inactive form such that it no longer has its function (s) in the microorganism can / can exercise.
• a functional inactivation of a gene can in particular also take place in that it is completely or partially replaced by an alternative gene.
• This alternative gene can then be expressed in place of the original gene.
• the originally present gene was functionally inactivated, instead the alternative gene is expressed, thereby causing the functional change.
• the alternative gene may be a gene related to the original gene (more than 50% identity to the original gene) or a gene unrelated to the original gene (50% or less identity to the original gene).
• the alternative gene can be inserted by insertion into the coding sequence of the original gene. This functionally inactivates the original gene and instead expresses the alternative gene.
• the genetic modification may be present both in the gene product coding sequence and in a gene regulatory sequence belonging to the gene.
• Microorganisms to be used according to the invention can be modified, for example, with regard to their requirements of the culture conditions, changed with regard to their movement behavior, altered with regard to their sporulation ability, altered with regard to a specific metabolic pathway - for example to prevent the formation of bad odors during the fermentation - or else other or have additional selection markers.
• genes in the Bacillus pumilus strain to be used in a method according to the invention are genetically modifiable, to which a correspondence exists in one or more of the genomes of the microorganisms indicated below: Agrobacterium radiobacter K84, Agrobacterium tumefaciens str. C58, Agrobacterium vitis S4, Arcobacter butzleri ED-1, Arcobacter nitrofigilis DSM 7299, Arcobacter sp. L, Aromatoleum aromaticum EbN1, Arthrobacter aurescens TC1, Arthrobacter chlorophenolicus A6, Arthrobacter phenanthrenivorans Sphe3, Arthrobacter sp.
• lactis V9 lactis V9, Bifidobacterium bifidum PRL2010, Bifidobacterium breve UCC2003, Bifidobacterium dentium Bd1, Bifidobacterium longum DJO10A, Bifidobacterium longum NCC27051, Bifidobacterium longum subsp. infantis ATCC 15697, Bradyrhizobium sp.
• Lactobacillus acidophilus 30SC Lactobacillus amylovorus GRL 1112, Lactobacillus brevis ATCC 367, Lactobacillus buchneri NRRL B-30929, Lactobacillus casei ATCC 334, Lactobacillus casei BD-II, Lactobacillus casei BL23, Lactobacillus casei LC2W, Lactobacillus crispatus ST10, Lactobacillus delbrueckii subsp.
• bulgaricus ND02 Lactobacillus fermentum CECT 5716, Lactobacillus gasseri ATCC 33323, Lactobacillus helveticus H10, Lactobacillus johnsonii NCC 533, Lactobacillus kefiranofaciens ZW3, Lactobacillus plantarum WCFS1, Lactobacillus reuteri SD2112, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus GG ATCC 53103, Lactobacillus ruminis ATCC 27782, Lactobacillus sakia subsp.
• DC3000 / Stenotrophomonas maltophilia K279a strain K279a, Streptobacillus moniliformis DSM 12112, Streptomyces avermitilis MA-4680, Streptomyces bingchenggensis BCW-1, Streptomyces cattleya NRRL 8057 main chromosomes, Streptomyces clavuligerus ATCC 27064, Streptomyces coelicolor, Streptomyces flavogriseus ATCC 33331, Streptomyces griseus subsp. griseus NBRC 13350, Streptomyces scabiei 87.22, Streptomyces sp.
• SirexAA-E Streptomyces venezuelae ATCC 10712, Streptomyces violaceus Tu 4113, Sulfobacillus acidophilus TPY, Thermobifida fusca YX, Thermotoga lettingae TMO, Thermotoga maritima MSB8, Thermotoga naphthophila RKU-10, Thermotoga neapolitana DSM 4359, Thermotoga petrophila RKU-1, Thermotoga sp.
• the modifiable genes in particular their sequences, are also available in publicly available databases, for example in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database at http://www.genome.jp/kegg or in US Pat NCBI (National Center for Biotechnology Information, U.S.A. National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894, U.S.A.) databases at http://www.ncbi.nlm.nih.gov.
• the KEGG database has been used since 1995 by the laboratories of Kanehisa et al. developed by the Kyoto University Bioinformatics Center and the Human Genome Center of the University of Tokyo.
• these databases also contain information and sequences of whole genomes or large parts of the genome of different microorganisms.
• a genetic correspondence in the sense of the present patent application is characterized on the one hand by the highest possible sequence homology between the gene of the Bacillus pumilus strain to be used according to the invention and the gene of Gioia et al. published Bacillus pumilus strain and / or the gene of the above microorganism.
• a genetic correspondence is characterized by a similar function, ie the mutually corresponding genes of the Bacillus pumilus strain to be used according to the invention and that of Gioia et al. Published Bacillus pumilus strain and / or the above microorganism have a similar function in the respective microorganism.
• the expert can determine the nucleic acid sequence with the highest sequence match in the genome of the Bacillus pumilus strain by genetic comparison and / or molecular biology standard method, which is genetically modified and then in the inventive method to be used.
• the confirmation of a similar function can provide comparative experiments with the respective microorganisms, in each of which the gene compared on the basis of the sequence comparison to the same Modified (preferably functionally inactivated) is and is observed whether similar changes occur in both microorganisms, in particular phenotypic changes. For example, does the change, in particular the functional inactivation, of the gene in question in the Gioia et al.
• Bacillus pumilus strain and / or in the microorganism mentioned above is accompanied by a change in metabolic activity, movement or sporulation behavior, and a corresponding change is observed in the Bacillus pumilus strain to be modified and used in accordance with the invention, this is an affirmation of the correct To see assignment.
• Appropriate methods are standard in the field of genetics, in particular the genetics of microorganisms, and are well known to those skilled in the art.
• the microorganism according to the invention is sporulation inhibited. This is preferably achieved by functionally inactivating its gene spolV (yqfD) or its genetic equivalent, in particular by deleting the gene spolV (yqfD) or its genetic equivalent or parts thereof. It has been found that with such a sporulation-inhibited Bacillus pumilus strain a particularly high product yield is achieved in a method according to the invention.
• the microorganisms used in the process according to the invention can be cultured and fermented in the usual way, for example in discontinuous or continuous systems.
• a suitable nutrient medium is inoculated with the microorganisms and the protein harvested from the medium after a period to be determined experimentally.
• Continuous fermentations are characterized by achieving a flow equilibrium, in which over a relatively long period of time cells partly die out but also regrow and at the same time the protein formed can be removed from the medium.
• Processes according to the invention are preferably fermentation processes. Fermentation processes are known per se from the prior art and represent the actual large-scale production step, usually followed by a suitable purification method of the protein produced. All fermentation processes which are based on a process according to the invention for producing a protein represent embodiments of a process according to the invention.
• Fermentation processes which are characterized in that the fermentation is carried out via a feed strategy, come in particular into consideration.
• the media components consumed by the ongoing cultivation are fed. This can result in significant increases in both cell density and cell mass or dry matter can be achieved.
• the fermentation can also be designed so that undesired metabolic products are filtered out or neutralized by the addition of buffer or suitable counterions.
• the produced protein can be harvested from the fermentation medium.
• a fermentation process is resistant to isolation of the protein from the microorganism, i. a product preparation from the cell mass (dry matter) is preferred. This can be achieved, for example, by the provision of suitable secretion markers or mechanisms and / or transport systems for the microorganisms to secrete the protein into the fermentation medium.
• the isolation of the protein from the host cell i. a purification of the same from the cell mass, carried out, for example by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.
• Microorganisms according to the invention are advantageously used in processes according to the invention for producing a protein. Consequently, a further subject of the invention is therefore the use of a microorganism according to the invention for the production of a protein, in particular of an enzyme.
• the transformed strains were used for fermentative protease production.
• the Bacillus licheniformis strain used is disclosed in the international patent application WO 91/02792 , As Bacillus pumilus strain Bacillus pumilus DSM 14395 was used in which the gene was spolV (yqfD) functionally inactivated by a deletion.
• the promoters used were nucleic acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2.
• the promoter is arranged in the respective expression plasmids in each case 5 'of the nucleic acid sequence which codes for the protease.
• the following plasmids were used (Table 1): Table 1: Plasmid No. promoter Protease gene 1 SEQ ID NO: 1 coding for variant F49 according to WO 95/23221 2 SEQ ID NO: 1 coding for variant F49 according to WO 95/23221 3 SEQ ID NO: 2 coding for variant F49 according to WO 95/23221
• the resulting production strains were used in a standard fermentation process in a 2 liter laboratory fermenter (48 h culture period) and the resulting protease activities were determined by the release of the chromophore para-nitroaniline (pNA) from the substrate sucrose.
• pNA chromophore para-nitroaniline
• AAPF L-Ala-L-Ala-L-Pro-L-Phe-p-Nitroanilide
• the protease cleaves the substrate and releases pNA.
• the release of pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of enzymatic activity (see Del Mar et al., 1979).
• the measurement is carried out at a temperature of 25 ° C, at pH 8.6, and a wavelength of 410 nm.
• the measuring time is 5 min and the measuring interval 20s to 60s.
• an expression plasmid As another expression plasmid (control) an expression plasmid was used, which differs from the plasmids 1 and 3 in that instead of a promoter from Bacillus pumilus a Bacillus licheniformis promoter was used, which is described in the international patent application WO 91/02792 ("promoter of the ATCC 53926 alkaline protease gene", see Examples 5, 6 and Figure 27) WO 91/02792 ). As Bacillus pumilus strain was used as in Example 1 Bacillus pumilus DSM 14395, in which the gene was spolV (yqfD) functionally inactivated by a deletion.
• This strain was transformed with the mentioned expression plasmids.
• the resulting production strains were used in a standard fermentation process in a 2 liter laboratory fermenter and the resulting protease activities determined by the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide (AAPF) as described in Example 1.
• pNA chromophore para-nitroaniline
• AAPF suc-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide
• Table 3 Plasmid No. relative protease activity (%) control 100 1 133 3 131
• Two different expression plasmids as indicated below, each comprising a gene encoding an amylase (target protein) and a functional promoter, were transformed into both a Bacillus licheniformis and a Bacillus pumilus strain .
• the transformed strains were used for fermentative amylase production.
• the Bacillus licheniformis strain used is disclosed in the international patent application WO 91/02792 , As Bacillus pumilus strain Bacillus pumilus DSM 14395 was used in which the gene was spolV (yqfD) functionally inactivated by a deletion.
• the promoters used were nucleic acid sequences according to SEQ ID NO: 3 and SEQ ID NO: 4 (amylase promoter from Bacillus amyloliquefaciens, as described in US Pat Palva, I., Pettersson, RF, Kalkkinen, N., Lehtovaara, P., Sarvas, M., Soderlund, H., Takkinen, K. and Kaariainen, L. "Nucleotide sequence of the promoter and NH2-terminal signal peptide region of the alpha-amylase gene from Bacillus amyloliquefaciens"; Gene 15 (1), 43-51 (1981 ) disclosed).
• the promoter is arranged in the respective expression plasmids in each case 5 'of the nucleic acid sequence which codes for the amylase.
• Table 4 Plasmid No. promoter Amylase gene 4 SEQ ID NO. 3 Coding for the protein according to Seq ID No: 2 EP1307547 A2 5 SEQ ID NO. 4 Coding for the protein according to Seq ID No: 2 EP1307547 A2 6 SEQ ID NO. 1 Coding for the protein according to Seq ID No: 2 EP1307547 A2 7 SEQ ID NO. 2 Coding for the protein according to Seq ID No: 2 EP1307547 A2
• the resulting production strains were placed in a 2 liter laboratory fermenter (48h culture period) in a standard fermentation process and the resulting amylase activities determined.
• a modified p-nitrophenyl maltoheptaoside was used terminal glucose unit is blocked by a benzylidene group; this is cleaved by amylase to free p-nitrophenyl oligosaccharide, which in turn is converted by means of the auxiliary enzymes glucoamylase and alpha-glucosidase to glucose and p-nitrophenol.
• the amount of released p-nitrophenol is proportional to the amylase activity.
• the measurement takes place, for example, with the Quick-Start® test kit from Abbott, Abott Park, Illinois, USA.
• the absorption increase (405 nm) in the test batch is detected at 37 ° C. for 3 minutes against a blank value by means of a photometer.
• the calibration is carried out via an enzyme standard of known activity (for example Maxamyl® / Purastar® 2900 the Genencor company with 2900 TAU / g).
• the evaluation is carried out by plotting the absorption difference dE (405 nm) per min against the enzyme concentration of the standard.
• Table 5 shows the relative measured amylase activities for Bacillus pumilus , which are related to plasmid 4 (promoter of SEQ ID NO: 3) amylase activity for Bacillus licheniformis, defined as 100%.
• Table 5 Plasmid No. relative amylase activity (%) in B. licheniformis relative amylase activity (%) in B. pumilus 4 100% 376% 5 Not determined 212%
• an amylase target protein
• Bacillus pumilus was examined with expression constructs comprising different promoters.
• the expression plasmids 4, 6 and 7 were used with promoters according to SEQ ID NO. 3, 1 and SEQ ID NO: 2 as described in Example 3.
• Bacillus pumilus strain was used as in Example 1 Bacillus pumilus DSM 14395, in which the gene was spolV (yqfD) functionally inactivated by a deletion.
• This strain was transformed with the mentioned expression plasmids.
• the resulting production strains were used in a standard fermentation process in a 2 liter laboratory fermenter and the resulting amylase activities determined as described in Example 3.

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